The present invention relates generally to new methods and systems for detecting, quantifying and monitoring extracellular or exogenously added hemoglobin, including hemoglobin substitutes, in a blood sample, particularly a whole blood sample, as well as in plasma and serum samples. The present invention further relates to the use of automated hematology analyzers to determine and quantify the concentration of extracellular and exogenous hemoglobin, including cell-free hemoglobin substitutes, in a blood, plasma, or serum sample, and is particularly advantageous for medical use during patient trauma or surgery, as well as for monitoring hemoglobin levels during recovery.
Whole blood substitutes have long been sought after as alternatives to whole blood for use in the medical field, particularly following trauma and/or surgery where transfusions are needed. Motivated by the need to supply large quantities of blood to the military on the battlefield, but limited by the resources to insure the safety of the blood supply in the face of contamination by human pathogens and viruses, notably hepatitis viruses and HIV, blood banks abandoned their whole blood supply programs in the early 1980s. However, the search for blood substitutes, e.g., synthetic blood substitutes, that are free of contaminants and that can be used in patient treatment has continued.
Currently, there is a renewed interest to produce and/or isolate a blood substitute. However, because of the complexity of blood and the various components that comprise whole blood, as well as the stringent federal regulations governing the testing and use of such synthetic products, industry has focused its research efforts on the development of products which temporarily deliver oxygen, rather than on the development of a variety of different products having other functions that transfused blood provides.
Hemoglobin (HGB) isolated from human or animal blood, or a synthetically produced oxygen carrier, such as perfluorocarbon, are two types of hemoglobin substitutes that are currently in clinical trials. Other red blood cell substitutes, i.e., oxygen-carrying hemoglobin substitutes, have also been developed and characterized for use in patients. (See, for example, Red Blood Cell Substitutes, 1998, (Eds.) A. S. Rudolph, R. Rabinovici, and G. Z. Feuerstein, Dekker, New York, N.Y.). Such oxygen-carrying hemoglobin substitutes may be used in conjunction with standard medical therapies, such as transfused blood or blood products.
As a specific but nonlimiting example, Enzon, Inc. (Piscataway, N.J.), has developed a polyethylene glycol (PEG)-modified bovine hemoglobin, abbreviated PEG-HGB. PEG-HGB is produced by a process in which strands of PEG are crosslinked to the surfaces of HGB molecules, for example, as disclosed in U.S. Pat. Nos. 5,386,014 and 5,234,903 to Nho et al.). Other specific, yet nonlimiting, examples include Hemopure(copyright) and Oxyglobin (Biopure, Cambridge, Mass.).
The first generation HGB substitutes were generally intended for short term treatment of blood/oxygen loss during surgery or following trauma. One disadvantage of HGB substitutes is the short circulation half-life attributed to these products. For example, HGB substitutes that are added to blood have a circulation half-life of up to 36 hours compared with a circulation half-life of up to 30 days for transfused blood. However, this relatively short half-life is typically not a serious problem associated with the use of such blood substitutes, because these products are predominantly indicated for short-term treatment objectives.
In determining whether or not to transfuse a patient who has a low blood hemoglobin concentration, the transfusion xe2x80x9ctriggerxe2x80x9d is between about 6 and 8 g/dL of hemoglobin in whole blood and depends on a number of specific factors, such as blood volume status, pulmonary, cardiac and cerebrovascular status, chronicity or severity of anemia, patient symptoms relating to blood loss, expected blood loss for a particular procedure, risk of re-bleeding from surgery, high risk patients (i.e., the elderly), and thrombocytopenia.
In general, the measurement of hemoglobin in whole blood samples is performed by commercially available automated hematology analyzers. To date, with the exception of certain hematology analyzers, such as those available from Bayer Corporation, e.g., the ADVIA 120(copyright) hematology analyzer system, other commercially-available blood analyzers are able to measure only total hemoglobin, which includes not only exogenously added hemoglobin, but also intracellular hemoglobin that is derived from the red blood cells in a blood sample. The present invention provides the ability to determine and measure exogenous hemoglobin in a whole blood, plasma, or serum sample in a reliable, reproducible and automated way.
It is an object of the present invention to provide automated methods and hematology systems to specifically and accurately detect, quantify and monitor different types of hemoglobin in a blood, plasma, or serum sample, preferably a whole blood sample, undergoing analysis, namely, (i) hemoglobin derived from red blood cells (i.e., intracellular hemoglobin, or cellular hemoglobin, as used herein); (ii) extracellular hemoglobin, or a hemoglobin product or substitute, particularly, a cell-free hemoglobin derivative, e.g., PEG-HGB, or a synthetic form of hemoglobin, e.g., Hemopure(copyright), (Biopure, Cambridge, Mass.); Oxyglobin, (Biopure, Cambridge, Mass.), which has been transfused into a patient requiring added HGB, or otherwise added to a blood, plasma, or serum sample (i.e., exogenous hemoglobin); and (iii) total hemoglobin (i.e., the combination of intracellular and exogenous hemoglobin).
It is another object of the present invention to provide the ability to monitor, during a course or regimen of treatment, hemoglobin or a hemoglobin product, derivative or substitute, such as a cell-free hemoglobin derivative, that has been added to blood of a patient or individual in need thereof. Also, in accordance with the present invention, hemoglobin, or a hemoglobin product, derivative or substitute, such as a cell-free hemoglobin derivative, can be monitored, determined or quantified as exogenous hemoglobin in a patient""s blood, plasma, or serum, after the patient has been transfused with such a hemoglobin product, or a substance containing the product (e.g., a physiologically acceptable solution or composition, and the like).
It is yet another object of the present invention to provide a system to differentiate and accurately measure the contribution of an added or exogenous hemoglobin product or blood substitute, e.g., PEG-HGB, separately and distinctly from the contribution of cellular HGB which derives from a patient""s red blood cells. In accordance with the present invention, the automated analytical method and system as described calculate a specific concentration of the extracellular hemoglobin in a blood sample which has been transfused with a hemoglobin product, or in a blood, plasma, or serum sample which contains extracellular hemoglobin to be detected. Thus, the invention allows the detection and monitoring of an extracellular hemoglobin component, even in the presence of a cellular hemoglobin component derived from the red blood cells in a given sample. In addition, through the present method, as little as about 0.5 g/dL of hemoglobin is detectable in a total of approximately 6.0 g/dL of extracellular hemoglobin. Experiments were performed in the HGB concentration range which is relevant to transfusions, i.e., a decision point of about 6-7 g/dL of total HGB in blood. Moreover, hemoglobin was recoverable and quantified in blood samples that were 24 hours old, and which had been stored at temperatures of 2xc2x0 C. to 8xc2x0 C.
Further objects and advantages afforded by the present invention will be apparent from the detailed description hereinbelow.
The present invention provides automated methods and hematology systems to specifically and accurately detect and quantify different types of hemoglobin in a whole blood, plasma, or serum sample. Automated hematology analyzers produced by and commercially available from Bayer Corporation, the assignee hereof, have been found to be able to directly determine and measure the concentration of exogenous, i.e., extracellular, hemoglobin in a sample. Suitable instruments for carrying out the analyses of the present invention possess two analytic channels which measure the concentration of hemoglobin in a blood sample. Specifically, and by way of example, the Bayer H*(trademark) series of hematology analyzer instruments and the Bayer ADVIA(copyright) series of hematology analyzer instrument systems (e.g., ADVIA 120(copyright)), as well as hematology analyzers with similar design or function, have the capability of performing quantitative analysis on the hemoglobin content of blood, plasma and serum which contain exogenous hemoglobin.
Other commercially available blood analyzers currently measure only total hemoglobin (i.e., the combination of cellular HGB and extracellular, exogenously added HGB) in a blood sample. However, the automated hematology instruments for use in the present invention, e.g., the above-mentioned Bayer hematology analyzers, are able to determine separately and independently the cellular HGB (reported as xe2x80x9cCalculated HGBxe2x80x9d), as well as total hemoglobin (reported as xe2x80x9cHGBxe2x80x9d) in a whole blood sample. To date, currently available hematology analyzers cannot simultaneously detect cellular hemoglobin and non-cellular hemoglobin, i.e., exogenously added hemoglobin, in a whole blood, plasma, or serum sample, and thus, cannot report the separate values of these measurements.
In addition, the monitoring of patient progress in those patients who have received exogenous hemoglobin, e.g., PEG-HGB, or a cell-free, oxygen-carrying hemoglobin substitute, such as Hemopure(copyright) or Oxyglobin (Biopure, Cambridge, Mass.) via transfusions, for example, is not possible with other commercially available analyzers, because these analyzers are not able to distinguish between the hemoglobin contributed by the exogenously provided HGB substitute and the hemoglobin contributed by the red blood cells in a whole blood sample, particularly in the cases of acute blood loss or autolysis.
In contrast to presently-used blood analyzers, the automated analyzers and methods according to the present invention can differentiate and accurately measure the contribution of an exogenous HGB substitute separately and distinctly from the contribution of cellular HGB derived from red blood cells. As described herein, the automated analyzers calculate a specific concentration of the extracellular hemoglobin in a whole blood, plasma, or serum sample which has been transfused with a hemoglobin product, thereby allowing the detection and monitoring of exogenous hemoglobin in the absence of the cellular hemoglobin component derived from the red blood cells in a given sample. In addition, the analyzers described herein provide cellular and total hemoglobin values for a blood sample containing an added hemoglobin product.
The present invention is particularly advantageous because a number of cell-free hemoglobin derivatives have been developed for use instead of whole blood, especially in trauma cases. Thus, the present invention provides a viable method for determining, measuring and monitoring levels of such hemoglobin products in blood, plasma or serum when such products have been added exogenously to blood, and introduced (e.g., transfused) into patients as a substitute for whole blood.
It will be appreciated that the method of the present invention embraces the analysis of blood samples, preferably whole blood samples, as well as plasma and serum samples, from patients who have received cell-free red blood cell substitutes, i.e., who have added hemoglobin or oxygen-carrying blood substitute products in their blood, for a variety of medical reasons. It will be further appreciated that there are a number of cell-free, hemoglobin-based red blood cell substitutes which can be added to blood, or used as blood substitutes, to treat patients requiring such red blood cell or oxygen-carrying blood substitutes, for various therapies and treatment conditions, such as transfusion, restoration of blood volume, treatment of acute blood loss, surgery, shock (e.g., hemorrhagic shock), or tumor oxygenation, for example.
Nonlimiting examples of cell-free, hemoglobin-based red blood cell substitutes, or oxygen-carrying substitutes, that can be determined, measured, and/or monitored in whole blood, plasma, or serum samples in accordance with the present methods include cross-linked, particularly chemically cross-linked, human hemoglobin products (e.g., D. J. Nelson, 1998, xe2x80x9cHemAssist: Development and Clinical Profilexe2x80x9d, In: Red Blood Cell Substitutes, 1998, (Eds.) A. S. Rudolph, R. Rabinovici, and G. Z. Feuerstein, Dekker, New York, N.Y., pp. 353-400; J. Adamson et al., 1998, Ibid., pp. 335-351; and T. M. S. Chang, 1998, Ibid., pp. 465-473); recombinant hemoglobin products, particularly recombinant human hemoglobin (e.g., J. H. Siegel et al., 1998, Ibid., pp. 119-164 and J. W. Freytag and D. Templeton, 1998, Ibid., pp. 325-222) or recombinant bovine hemoglobin; purified, preferably ultrapurified, animal hemoglobin products, e.g., ultrapurified bovine hemoglobin and ultrapurified human hemoglobin; and animal-based oxygen-carrying products, for example, bovine hemoglobin-based oxygen carrier (HBOC) products, e.g., xe2x80x9cHemopure(copyright)xe2x80x9d and oxyglobin (Biopure, Cambridge, Mass.); (W. R. Light et al., 1998, Ibid., pp. 421-436; T. Standl et al., 1998, Br. J. Anaesth., 80(2):189-194; and Palaparthy et al., 2000, Adv. Drug Delivery Reviews, 40:185-198). The purified or ultrapurified hemoglobin products are cell-free and can be cross-linked or polymerized, e.g., Hemopure(copyright) and Oxyglobin, (Biopure, Cambridge, Mass.).
The use of automated hematology analyzers in the methods according to the present invention provides further advantages, which are described herein and demonstrated by the Examples as set forth below. More specifically, and by way of example, the use of automated hematology analyzer analysis according to this invention allows the detection and measurement of extracellular (or non cell-derived) PEG-HGB (e.g., xe2x89xa70.2 to 5.6 g/dL of blood) that is added to anticoagulated whole blood samples. The recovery of PEG-HGB is linear (e.g., xe2x89xa70.2 to 5.6 g/dL of blood) when added to plasma or whole blood samples. Also, PEG-HGB is recoverable in 24 hour old samples that have been stored at 2-8xc2x0 C. In addition, the bovine hemoglobin products, Hemopure (Biopure, Cambridge, Mass.) and Oxyglobin, also obtained from Biopure, have been added to whole blood and plasma samples and accurately detected as oxygen-carrying blood substitutes according to the methods of the present invention. (Examples 5 and 6).
The added HGB component in a blood sample is obtained by determining the difference between the total HGB (computed from the calorimetric absorbance in the hemoglobin channel of the hematology analyzer) and the calculated cellular HGB (derived from the red blood cell (RBC) cytogram in the red cell channel of the hematology analyzer), which is calculated by the formula: (RBCxc3x97MCVxc3x97CHCM/1000), where MCV is mean cell volume and CHCM is Cellular Hemoglobin Concentration Mean, which measures the same cellular property as MCHC, or Mean Cellular Hemoglobin Concentration, in unlysed blood. The CHCM value is obtained from the Red Blood Cell channel of the hematology analyzer, such as the ADVIA 120(copyright) hematology system.
In particular, CHCM is obtained from light scattering measurements according to Mie Theory (see Tycko et al., 1985, Appl. Optics, 24:1355-1365, and U.S. Pat. No. 4,735,504 to Tycko). In contrast, MCHC is obtained by dividing total HGB by the product (MCVxc3x97RBC). Practically speaking, MCHC is not exactly equal to CHCM for normal blood samples, but these values preferably agree closely. For example, the MCHC value associated with the added hemoglobin component is preferably within a range of about 0-5 g/dL of blood, more preferably between about 0-2 g/dL of blood, of the CHCM value. The difference between total hemoglobin and intracellular hemoglobin is termed xe2x80x9cHGB Deltaxe2x80x9d (xe2x80x9cHGBxcex94xe2x80x9d) and represents the concentration of exogenously added hemoglobin, e.g., PEG-HGB, in a blood sample.
An explanation related to the above-mentioned lack of complete equality between the MCHC and CHCM values is as follows. After a typical meal, for example, it is not uncommon for the blood plasma to develop a small degree of lipemia (i.e., a suspension of small submicroscopic and microscopic particles of lipids, called chilomicrons). The presence of the particles causes a minor amount of light scattering, thereby diminishing the amount of light transmitted through a solution of hemoglobin in a hemoglobinometer. Consequently, the solution appears to contain slightly more hemoglobin than it actually does. The cell by cell measurements of hemoglobin concentration, performed by the Bayer ADVIA 120(copyright) hematology analyzer, are free of this error. The ADVIA 120(copyright) is calibrated such that if xcex94HGB is greater than 1.9 gm/dL, a sample is flagged as abnormal; i.e., a degree of lipemia in excess of this amount is considered abnormal. Also, if part of the blood sample has hemolysed, either in vivo in the patient or in the collection tube, a xcex94HGB value is also produced. The two HGB measurements performed by the ADVIA 120(copyright) analyzer alert the physician or clinician to the existence of any abnormal lipemia or hemolysis in a patient sample.
Until the present invention, no other commercially available automated hematology analyzer was able to detect simultaneously the intracellular (or cellular) hemoglobin (xe2x80x9cCalculated HGBxe2x80x9d) and extracellular HGB (HGBxcex94) in a whole blood sample. Accordingly, the present invention provides the ability to monitor added hemoglobin substitutes to blood by determining the amount of added hemoglobin substitute independently of the hemoglobin contributed by the red blood cell component of blood. For normal unlysed, and for abnormal blood samples, with a properly calibrated system, HGB Delta equals zero.
In accordance with an embodiment of the present invention, the hematology analyzers suitable for use in the present invention, e.g., Bayer ADVIA 120(copyright) and the Bayer H*(trademark) System series of hematology analyzers, are able to directly measure the concentration of exogenous extracellular hemoglobin because these instruments possess two analytic or detection channels, each of which measures a different type of hemoglobin concentration in a whole blood or plasma sample.
In such instruments, one of the analytic or detection channels is the Hemoglobin (HGB) channel which measures the concentration of total hemoglobin in the sample by means of hemolysis and extraction of the hemes from their biological complex with globin, forming a ligated ferric heme species which is captured in a surfactant micelle and is measured spectrophotometrically (See, for example, U.S. Pat. No. 5,858,794 to M. Malin; M. Malin et al., 1992, Anal. Chim. Acta, 262:67-77; and M. Malin et al., 1989, Am. J. Clin. Path., 92:286-294). The second analytic or detection channel in such instruments is the Red Blood Cell (RBC) channel which measures the red blood cell concentration and the mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC) of approximately 10,000 individual erythrocytes as they pass through two light scattering detectors.
The presence and design of hematology analyzers having both an HGB channel and an RBC channel, in conjunction with two light scattering detectors which detect the light scattered on a cell-by-cell basis as a blood sample containing RBCs passes through the RBC optical channel, allow a difference between intracellular hemoglobin and extracellular hemoglobin to be determined and calculated, thereby providing the performance of the method described herein. For a description of the optical mechanisms of suitable automated analyzers that are capable of performing the method of the present invention, see Kim and Ornstein, 1983, Cytometry, 3:419-427; U.S. Pat. No. 4,412,004 to Ornstein and Kim; Tycko et al., 1985, Appl. Optics, 24:1355-1365; U.S. Pat. No. 4,735,504 to Tycko; and Mohandas et al., 1986, Blood, 68:506-513.
As a specific but nonlimiting example, the Bayer ADVIA 120(copyright) hematology analyzer is capable of calculating the difference (xe2x80x9cHGB Deltaxe2x80x9d, or xe2x80x9cHGBxcex94xe2x80x9d) between the total and intracellular HGB concentrations (all HGB concentrations are in grams per deciliter, g/dL, of whole blood), as follows:
HGBxcex94, g/dL=Total HGB, g/dLHGB Channelxe2x88x92Intracellular HGB, g/dLRed Cell Channel.
In the above equation, HGBxcex94 represents the concentration of the extracellular HGB in the blood, plasma, or serum sample. Under ordinary conditions, delta HGB (HGBxcex94)=0.
Thus, according to the present invention, total hemoglobin is measured and monitored using the HGB channel of the hematology instrument, while the RBC channel detects only the intracellular HGB contained within the red blood cells of a blood sample. These two measurements are subtracted to yield the HGB Delta, which represents extracellular HGB.
In the system according to the present invention, HGB delta was introduced as a readout parameter in order to provide a check on the HGB results obtained in the HGB channel with certain types of abnormal or pathological blood samples, including lipemic and icteric blood samples and samples having elevated white blood cell counts. Such abnormal blood samples have been shown to yield artificially elevated HGB results in the H*(trademark) System HGB channel (See, M. Malin et al., 1989, Am. J. Clin. Path., 92:286-294). For example, certain blood samples scatter light, and such light scattering interference causes some of the light to be undetected. This light scattering interference causes an apparent increase, or artificial elevation, in the absorbed light, or absorbance value, for the sample. Because an HGB concentration can be obtained from the Red Blood Cell channel of a hematology analyzer (Tycko et al., 1985, Appl. Optics, 24:1355-1365), HGBxcex94 (or HGB Delta) was newly found by the present inventors to be able to be introduced as a readout parameter for the analyzer, wherein the value for intracellular hemoglobin derived from the analyzer""s Red Cell channel was subtracted from the value for total hemoglobin derived from the analyzer""s Hemoglobin channel to yield the HGBxcex94 value (g/dL in blood).
The present method of measuring and determining the intracellular versus extracellular, or exogenously added, HGB concentration in a whole blood sample, as well as the total HGB concentration, is capable of being used and performed on any of the commercially available Bayer H*(trademark)System or ADVIA 120(copyright) hematology analyzer instruments. However, it will be understood by those having skill in the pertinent art that other hematology instruments having a two channel system of measuring HGB concentration in the blood can be designed to perform the method of HGB determination and monitoring as described herein, and are embraced by the present invention. Also embraced by the present method are a series or combination of hematology analyzers which are designed and/or programmed to operate on the basis of a two channel hemoglobin analysis system.
Thus, the present invention provides a method for directly determining and monitoring extracellular hemoglobin concentration in a blood sample, preferably a whole blood sample, that is performed on an automated hematology analyzer, preferably having two analytic channels for hemoglobin measurement. The method involves the determination of the total hemoglobin concentration of a blood sample aliquot in a channel of the analyzer that is suitable for determining detecting, and/or measuring hemoglobin. By way of the present method, total hemoglobin concentrations of about 0.5-1 g/dL, or 1.1 g/dL, to about 25 g/dL of blood, preferably about 1 g/dL to about 25 g/dL, more preferably about 2 g/dL to about 25 g/dL of blood, and most preferably, about 6 g/dL to about 22 g/dL of blood are able to be determined in a blood, plasma, or serum sample, or aliquot thereof. As will be appreciated by the skilled practitioner, a normal hemoglobin value is in the range of about 11-18 g/dL of blood. For females, the normal hemoglobin range is about 11-16 g/dL of blood; for males, the normal hemoglobin range is about 13-18 g/dL of blood; and for newborns, the normal hemoglobin range is about 16-20 g/dL of blood (Fundamentals of Clinical Chemistry, Eds. N. Tietz, W. B. Saunders Co., 1970, p. 944). Also, by way of example, an anemic individual would typically be likely to have a hemoglobin value in the range of about 6 to about 12 g/dL of blood.
The method further involves the determination of the intracellular concentration of the blood sample aliquot in a channel of the analyzer that is suitable for determining, detecting and/or measuring red blood cells. Intracellular hemoglobin amounts that are able to be determined and measured by way of the present method include HGB concentrations of from about 0.5-1 g/dL, or 1.1 g/dL, to about 25 g/dL of blood, preferably about 1 g/dL to about 25 g/dL, more preferably about 4 g/dL to about 24 g/dL of blood and most preferably, about 5 g/dL to about 23 g/dL of blood. A most preferred range of HGB concentrations for detection is about 6 g/dL to about 18 g/dL of blood. A critical total hemoglobin concentration that is able to be measured by the method of this invention is about 6 g/dL, more preferably, 5.6 g/dL, which are concentrations of hemoglobin that are relevant to transfusions, where the decision point for transfusing a patient is approximately 6-7 g/dL of total hemoglobin.
When the total and intracellular hemoglobin concentrations have been determined, these values are used to calculate the difference between total and intracellular hemoglobin concentrations so as to arrive at the value for the extracellular hemoglobin concentration in the blood sample, a value which is calculated automatically by the hematology analyzer. According to the present invention, the red cell channel of the hematology analyzer measures the hemoglobin concentration in whole blood as follows:
[HGB]Blood, Red Cell Channel/Intracellular (g/dL)=[CHCM (g/dL)xc3x97RBC Count (cells/mm3)xc3x97MCV (femtoliters/cell)/1000].
The HGB channel measures that total hemoglobin concentration, i.e., [HGB]Intracellular+[HGB]Extracellular.
In accordance with the described method, the automated hematology analyzer, e.g., ADVIA(copyright)120, calculates the difference between the Total HGB concentration and Intracellular HGB concentration to yield the HGB Delta, which corresponds to the extracellular or exogenous HGB concentration.